Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

Linnane, E., Davey, P., Zhang, P., Puri, S., Edbrooke, M., Chiarparin, E., Revenko, A. S., Macleod, A. R., Norman, J. C. and Ross, S. J. (2019) Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines. Nucleic Acids Research, 47(9), pp. 4375-4392. (doi: 10.1093/nar/gkz214) (PMID:30927008) (PMCID:PMC6511877)

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Abstract

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

Item Type:Articles
Additional Information:This study was funded by AstraZeneca.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Norman, Professor James
Authors: Linnane, E., Davey, P., Zhang, P., Puri, S., Edbrooke, M., Chiarparin, E., Revenko, A. S., Macleod, A. R., Norman, J. C., and Ross, S. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in Nucleic Acids Research 47(9):4375-4392
Publisher Policy:Reproduced under a Creative Commons License

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